The overall aim will be to examine in detail the physiology and pathophysiology of the metabolic regulation of adipose tissue lipoprotein lipase (ATLPL). ATLPL is not only important in the physiological removal of triglyceride from plasma lipoproteins, but alterations in ATLPL are seen in common metabolic disease states such as uncontrolled diabetes and obesity. In vitro, both cultured isolated rat and human adipocytes will be used to probe the molecular mechanism of LPL regulation. Initial studies will involve a purification of rat adipocyte LPL which will be used to generate monoclonal antibodies against the protein. By radioisotopically labeling LPL in cultured rat adipocytes, monoclonal antibodies will be used to immunoprecipitate LPL so that it can be visualized by autoradiography on sodium dodecyl sulfate polyacrylamide gels. Using this tool, the effects of glucose and insulin on LPL transcription, translation and post-translational modification will be examined to determine the molecular level of their effects. Several rat models of obesity will be used to determine if increased enzyme synthesis is the mechanism for the increased amount of LPL activity per fat cell in the obese state. In addition, studies in cells prepared from diabetic rats will attempt to localize the molecular defect in insulin, glucose regulation of LPL seen in diabetes. Cultured isolated human adipocytes will be used to test the relationship of LPL measured in adipose tissue pieces to that measured in the adipocytes. Methods to perfect the culture of adipocytes from obese patients will be emphasized. This will be important to determine if increases in ATLPL/cell in obesity are secondary to intracellular or extracellular increases in enzyme. The relationship between LPL bioactivity and enzyme protein will also be characterized utilizing monoclonal antibodies against human LPL. In vivo studies will examine if regional differences exist in fasting and/or insulin-stimulated ATLPL in obese subjects. Such differences could relate to the normal or excessive distribution of body fat. In addition, sensitivity of the ATLPL response to insulin will be tested at low insulin concentrations (approximately 35 Muu/ml), to determine if differences exist between normal weight and obese subjects. Studies of ATLPL regulation before and after weight loss will examine if increases in responsiveness predict the resumptiom of the formerly obese state. Both the euglycemic clamp and meal-associated regulatory studies will be used here. Overall, these studies will combine an in vivo approach to establish a deeper understanding of the relevance of LPL regulation in the adipocyte and in adipose tissue to human physiology and diseases such as obesity and diabetes mellitus.